The long-term goal of this project is to define the molecular mechanisms which regulate epigenome stability during aging and the role of the mammalian sirtuin, SIRT6, in this process. Growing evidence indicates that epigenome structure becomes compromised with age which may be the root cause of age-related decline in cell and organ function. SIRT6 emerged as a critical regulator of multiple pathways related to aging such as genome and epigenome stability, tumorigenesis, inflammation and glycolysis. Additionally, SIRT6 overexpression extends the lifespan of mice. Our laboratory has demonstrated that SIRT6 is an upstream regulator of DNA double strand break (DSB) repair. We demonstrated that SIRT6 is phosphorylated by JNK1/2 in response to oxidative stress on amino acid S10 and this phosphorylation is required for the stimulation of DSB repair. We have shown that, in addition to controlling DSB repair, SIRT6 maintains genome stability by repressing transposable elements, and that oxidative stress causes re-localization of SIRT6 from the promoters of transposable elements to the DNA breaks. SIRT6 has two biochemical activities deacetylase (deacetylase) and mono-ADP ribosylase. The function of SIRT6 deacetylase activity is best characterized. In contrast, mono-ADP ribosylation activity of SIRT6 is much less studied. Our work has implicated this activity in DNA repair and epigenome stability. Our recent unpublished data analyzing biochemical functions of a SIRT6 variant found in centenarians, showed that centenarian SIRT6 has reduced deacetylation activity and enhanced mono-ADP ribosylation activity. This suggests that SIRT6 mono-ADP ribosylation activity is important for longevity. Therefore, we set out to identify the function and new targets of SIRT6 mono-ADP ribosylation. Using mass spectrometry we identified novel targets for SIRT6 mono- ADP ribosylation including histone H1, H2A, H2A.J, and chromatin remodelers BRG1 and SMARCC2. Furthermore, we showed that SIRT6-mediated ribosylation of SMARCC2 is required for activation of Nrf2 target genes in response to oxidative stress. Thus, we are ideally positioned to conduct further mechanistic studies of the role of SIRT6 mono-ADP ribosylation activity in epigenome stability. We will pursue the following specific aims: (1) examine the role of SIRT6 in maintaining epigenome stability in the context of cellular senescence and aging; (2) examine the mechanisms of SIRT6 effect on epigenome; specifically, the biological function of H1, H2A, H2A.J, BRG1 and SMARCC2 mono-ADP ribosylation; and (3) determine the role of SIRT6 mono-ADP ribosylation activity in epigenome stability and longevity by analyzing the knock-in mouse model which expresses ribosylation deficient SIRT6 mutation. The proposed research will delineate new pathways regulated by SIRT6, which are relevant to epigenome stability and aging. As such, we expect that these experiments will reveal critical, new information about the aging process, and will help to develop new strategies for treating age-related diseases.